Identification, tissue expression, and glucocorticoid responsiveness of alternative first exons of the human glucocorticoid receptor.

Transcripts for the human glucocorticoid receptor (NR3C1) are known to contain alternative first exons 1A1, 1A2, and 1A3 from the distal promoter or 1D, 1E, 1B, 1F, 1C, or 1H from the proximal promoter. Here, we report two additional alternative first exons identified by Rapid amplification of cDNA ends (RACE)-PCR. The first, exon 1I, starts approximately 700 bp downstream of the splice donor site of the longest form of exon 1A, 1A3, considerably extending the known distal promoter region with a region containing conserved transcription factor-binding sites as well as a potential glucocorticoid response element (GRE) that differs from the consensus GRE in only two positions. The second, exon 1J, is part of the proximal promoter region and resides between exons 1D and 1E. Since this has been determined by quantitative real-time reverse transcriptase (RT)-PCR, exon 1I is used foremost in cells of the T-lymphocyte lineage. In the T-ALL cell line CEM-C7H2, which is sensitive to glucocorticoid-induced apoptosis, transcripts containing alternative first exons from the distal as well as the proximal promoter regions were markedly autoinduced by glucocorticoid treatment, with more pronounced relative induction in the distal promoter. Neither transcript was autoinduced in the related, resistant cell lines CEM-C1, and CEM-C7R5. In contrast, the glucocorticoid-sensitive PreB697 cell line strongly autoinduced transcripts from the proximal promoter, but not transcripts from the distal promoter, to relevant levels. Therefore, the autoinductive feedback loop implicated in glucocorticoid-induced apoptosis cannot universally rely on the distal promoter of the glucocorticoid receptor.

Glucocorticoid-induced apoptosis and glucocorticoid resistance in acute lymphoblastic leukemia.

Glucocorticoids (GC) induce cell cycle arrest and apoptosis in lymphoid cells, and therefore constitute a central component in the treatment of lymphoid malignancies, particularly childhood acute lymphoblastic leukemia (ALL). In spite of its clinical significance and considerable efforts in many laboratories, however, the molecular basis of GC-induced apoptosis and the clinically important resistance phenomenon remains poorly defined. The anti-leukemic GC effects are critically dependent upon sufficient expression of the GC receptor (GR) throughout the response. In ALL cell lines, this is associated with, and may depend upon, GR autoinduction. In corresponding in vitro models, GC resistance frequently results from mutations in the GR gene and/or deficient regulation of its expression. The downstream components of the pathway, i.e., the GC-regulated genes responsible for cell death induction, have been studied by microarray-based comparative expression profiling, resulting in identification of a considerable number of GC-regulated candidate genes. Their possible function in the death response is, however, still undefined. One model predicts direct regulation of the apoptotic machinery, e.g., components of the "Bcl-2 rheostat", while a complementary hypothesis suggests deleterious GC effects on essential cellular functions, such as metabolism, production of and/or response to oxygen radicals, general transcription/translation, pH and volume control, etc. These regulatory effects may entail cell death, particularly if maintained for sufficient time through GR autoinduction. The latter form of cell death may occur even in the absence of functional apoptotic machinery (e.g., when caspases are blocked), but in this case appears to entail a more necrotic morphology. Taken together, GC may induce different types of cell death through distinct molecular pathways, depending on the cellular context. GC resistance might frequently result from defective GR expression, perhaps the most efficient means to target multiple antileukemic pathways.

Identification of glucocorticoid-response genes in children with acute lymphoblastic leukemia.

The ability of glucocorticoids (GCs) to kill lymphoid cells led to their inclusion in essentially all chemotherapy protocols for lymphoid malignancies, particularly childhood acute lymphoblastic leukemia (ALL). GCs mediate apoptosis via their cognate receptor and subsequent alterations in gene expression. Previous investigations, including expression profiling studies with subgenome microarrays in model systems, have led to a number of attractive, but conflicting, hypotheses that have never been tested in a clinical setting. Here, we present a comparative whole-genome expression profiling approach using lymphoblasts (purified at 3 time points) from 13 GC-sensitive children undergoing therapy for ALL. For comparisons, expression profiles were generated from an adult patient with ALL, peripheral blood lymphocytes from GC-exposed healthy donors, GC-sensitive and -resistant ALL cell lines, and mouse thymocytes treated with GCs in vivo and in vitro. This generated an essentially complete list of GC-regulated candidate genes in clinical settings and experimental systems, allowing immediate analysis of any gene for its potential significance to GC-induced apoptosis. Our analysis argued against most of the model-based hypotheses and instead identified a small number of novel candidate genes, including PFKFB2, a key regulator of glucose metabolism; ZBTB16, a putative transcription factor; and SNF1LK, a protein kinase implicated in cell-cycle regulation.